Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs

Fat deposition is a prominent economic trait and complex process in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Histological analysis of backfat tissue showed that adipocyte area increased and adipocyte number decreased with the development of backfat tissue in different stages (60d, 120d, 180d, 240d, 300d, and 360d). Meanwhile, a total of 1024 differentially expressed genes were identified in five comparison groups (120d vs 60d, 180d vs 120d, 240d vs 180d, 300d vs 240d, and 360d vs 300d). And then strongly co-expressed genes in modules were clustered by Weighted gene co-expression network analysis (WGCNA). Among these modules, turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had close correlation with 60d, 120d, 180d, 240d, 300d, and 360d developmental stage, respectively, while tan, black and turquoise module had intimate relation with backfat thickness, adipocyte area and adipocyte number, respectively. Furthermore, GO and KEGG enrichment were analyzed in each module, the gene co-expression network was constructed, and the hub genes were described. According to the co-expression network, 13 genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized as hub genes. Further analysis revealed that ACSL1 and ACOX1 might perform considerable function in early developmental stage of backfat tissue before 60d. FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position around 120d developmental stage. Besides, the functional relevance associated with SELP and backfat thickness, DNM1 and adipocyte area needs to be elucidated. In addition, QCR analysis validated the strong consistency of genes expressions with RNA-seq data, manifesting the reliability and accuracy of our study. The present research preliminarily depicted the gene co-expression network for each period of backfat developmental stage in Ningxiang pigs, which not only help to understand the integrated mechanism underlying backfat tissue development and lipid metabolism, but also promotes the progress of genetic improvement in pigs. Overall design: Eighteen castrated Ningxiang pigs in six developmental stages (60d, 120d, 180d, 240d, 300d and 360d) were used in this study. Six groups of pigs were half-sibs, and the three samples in each developmental stage were full-sibs. All the experimental pigs were reared under the standard environmental conditions. The backfat tissue at the position of the thickest point in shoulder were collected, and the sample was immediately frozen in liquid nitrogen for the further RNA-sequencing.

脂肪沉积是猪重要的经济性状，同时也是一个复杂的生物学过程。宁乡猪（Ningxiang pig）作为中国四大地方良种之一，以高脂肪沉积能力为典型特征。目前对于宁乡猪背膘组织不同发育阶段的基因表达调控模式仍不明晰，本研究旨在探究宁乡猪背膘组织发育的潜在分子调控因子。
对背膘组织的组织学分析显示，在60日龄、120日龄、180日龄、240日龄、300日龄及360日龄这几个发育阶段中，随着背膘组织的成熟，脂肪细胞面积逐渐增大，而脂肪细胞数量则逐步减少。
同时，在5组对比组合（120日龄vs60日龄、180日龄vs120日龄、240日龄vs180日龄、300日龄vs240日龄及360日龄vs300日龄）中共鉴定出1024个差异表达基因。
随后，通过加权基因共表达网络分析（Weighted gene co-expression network analysis, WGCNA）对模块内的强共表达基因进行聚类。在这些模块中，绿松石模块、红色模块、粉色模块、浅绿松石模块、深橙色模块以及深绿色模块分别与60日龄、120日龄、180日龄、240日龄、300日龄及360日龄发育阶段呈显著相关；而棕褐色模块、黑色模块及绿松石模块则分别与背膘厚度、脂肪细胞面积及脂肪细胞数量紧密关联。
此外，本研究对每个模块进行了基因本体（Gene Ontology, GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析，构建了基因共表达网络，并鉴定了核心基因（hub genes）。基于该共表达网络，最终鉴定出13个核心基因：ACSL1、ACOX1、FN1、DCN、CHST13、COL1A1、COL1A2、COL6A3、COL5A1、COL14A1、OAZ3、DNM1及SELP。
进一步分析表明，ACSL1与ACOX1可能在60日龄前的背膘组织早期发育阶段发挥重要调控作用；FN1、DCN、COL1A1、COL1A2、COL5A1、COL6A3及COL14A1则在120日龄发育阶段具有不可忽视的调控地位。此外，SELP与背膘厚度、DNM1与脂肪细胞面积之间的功能关联仍有待阐明。
另外，QCR分析验证了基因表达量与RNA测序（RNA-sequencing）数据的高度一致性，证实了本研究的可靠性与准确性。本研究初步绘制了宁乡猪背膘组织各发育阶段的基因共表达网络，不仅有助于解析背膘组织发育与脂质代谢的整体调控机制，同时也可为猪的遗传改良工作提供理论支撑。
试验设计：本研究共选取18头阉割后的宁乡猪，涵盖6个发育阶段（60日龄、120日龄、180日龄、240日龄、300日龄及360日龄）。6组试验猪两两之间为半同胞关系，且每个发育阶段的3个样本均为全同胞个体。所有试验猪均在标准饲养环境条件下进行饲养。采集肩胛部最厚处的背膘组织样本，随后立即将样本置于液氮中速冻，以备后续RNA测序（RNA-sequencing）使用。